Synthetic biology is a new approach for design and construction of new biological systems, and for re-design of natural biological systems. To pursue large-scale genomic engineering of a cell, an efficient high-throughput method that can simultaneously introduce many genes into a genome is required.
Various techniques have been developed to enable the assembly of several genes or DNA is modules into larger constructs, such as chain reaction cloning (Pachuk et al., 2000, Gene 243:19-25), ordered gene assembly in Bacillus subtilis (OGAB) (Tsuge et al., 2003, Nucleic Acid Res 31:8), DNA assembler in vivo (Shao et al., 2004, Nucleic Acid Res 37:10), uracil-specific excision reagent (USER) cloning (Bitinaite et, al., 2007, Nucleic Acids Res 35: 1992-2002), mating-assisted genetically integrated cloning (MAGIC) (Li and Elledge, 2005, Nat Genet 37: 311-319), sequence- and ligation-independent cloning (SLIC) (Li and Elledge, 2007, Nat Methods 4: 251-256), In-Fusion (Clontech; Marsischky and LaBaer, 2004, Genome Res 14: 2020-2028), polymerase incomplete primer extension (PIPE) (Klock et al., 2008, Proteins 71: 982-994), circular polymerase extension cloning (Quan and Tian, 2009 PLOS-One 4:6), and one-step assembly in yeast (Gibson et al., 2008, Proc Natl Acad Sci 105(51):20404-9; Gibson et al., 2009, Nat Methods 6:341-345). These methodologies are based mainly on historically well-characterized hosts, such as Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae. Other less-studied organisms are still in need of molecular biological tools.
A high demand for cellulosic biofuel is expected in the future. The hydrolysis of cellulose can be better catalyzed by a combination of many cellulases, including endo-β-1,4-glucanases, cellobiohydrolases, cellodextrinases, and exoglucosidases, as the synergism of multiple enzymes can enhance cellulolytic efficiency. One strategy is to engineer yeast strains for producing cocktails of enzymes.